The ability to perform chemical measurements in microscopic volumes has many applications in analytical chemistry. This is particularly true for the broad group of heterogeneous “ligand binding assays.” This broad class includes hybridization assays for specific DNA sequences, immunoassays employing immobilized antigen or antibody, and the receptor assays used in high throughput screening of pharmaceuticals.
When such assays are “scaled down” to the microscopic level, there is a clear reduction in the cost per test, related to the use of smaller quantities of the “sorbent” materials—the specific antibodies, nucleic acid probes, or receptor molecules employed. Because these reagents must often have exquisite selectivity for the analyte, while remaining inert to other sample constituents, they often represent a large fraction of the total reagent cost in conventional assay methods. In addition to the scale-related reduction of cost, micro-scale methods may facilitate parallel testing for multiple analytes in a single sample. Scaling down permits multiple tests to be probed optically within the constraints of conventional optical systems.
The theory of microscopic assay has been advanced by the work of Ekins et al. {“Multianalyte Microspot Immunoassay—Microanalytical “Compact Disc” of the Future,” Clin. Chem 37(11), 1955–1967 (1991); “Development of Microspot Multi-Analyte Ratiometric Immunoassay using Dual Fluorescent-Labelled Antibodies,” Anal. Chim. Act. 227, 73–96 (1989)}, which has focused on the concept of “ambient analyte” assay. Under ambient analyte conditions, the equilibrium number of analyte molecule bound by the solid phase is minimal relative to the number of analyte molecules present in solution. Such a regime minimally perturbs the analyte concentration in the solution over the solid phase during the course of an assay. This approach has the theoretical advantage of perturbing minimally any equilibria between the analyte and other sample constituents. For example, in a diagnostic assay it may be desirable to determine the concentration of a “free” drug molecule in the presence of an albumin-bound form without pertubing the physiological equilibrium of the sample. The “ambient analyte” assay also has the advantage of requiring minimal mass transport from solution to the surface, and so makes minimal demands on the kinetics of transport from bulk solution to a microscopic region bearing capture reagent.
Ambient analyte assays have been modeled by Ekins et al. using the mass action law and chemical activities computed by dividing the amount of analyte binding sites provided on the solid support into the total liquid reagent volume. With these assumptions, “ambient analyte assay” conditions are said to exist if the binding site “activity” is <0.01 K−1, where K is the ligand binding affinity in liter mole−1. Ekins et al. point out that reducing the area of the solid phase employed in an assay reduces also the background signals, which often limit the sensitivity of ligand binding assays. Although the “ambient analyte” approach tends to reduce signal by reducing the amount of analyte bound, the background reduction afforded by the use of microscopic solid phase regions permits the overall signal to background ratio to remain favorable to high sensitivity.
The ambient analyte regime, by virtue of its low percent recovery of analyte, makes stringent demands on laser monochromaticity, optical filters, permissible stray light, and perhaps, most importantly, the permissible background from the solid phase itself. Moreover, the practical concerns of analyte concentration and sample volume limits the total number of analyte molecules available for detection. Indeed, for some micro-scale applications, the entire amount of analyte available in the sample may be barely sufficient for practical detection above background.
For the foregoing reasons there is a need for microscale assay methods having increased sensitivity for very low quantities of analyte. Preferably, the miniaturized binding assays will maximize the amount of analyte bound to a substrate for detection and quantification. Moreover, such scaled down binding assays should minimize the use of costly “sorbent materials’ and permit more than one assay to be performed on the same sample volume.